Cryptosporidiosis is predominantly a gastrointestinal disease of humans and other animals, caused by various species of protozoan parasites representing the genus Cryptosporidium. Detection of Cryptosporidium spp. in human clinical samples is central to the prevention, surveillance and control of cryptosporidiosis, particularly given that there is presently no broadly applicable treatment regimen for this disease. A non-radioactive, genus specific DNA dot blot hybridization assay was developed using Digoxigenin (DIG) labelled probes to detect Cryptosporidium DNA in human clinical samples. Four hundred fifty (n = 450) clinical samples were subjected to microscopic examination, Polymerase Chain Reaction assay (PCR), Dot blot hybridization assay and Real Time PCR assay. A total of forty-one (n = 41) samples were positive by microscopy, forty-two (n = 42) by both PCR assay and dot blot hybridization assay and forty-three (n = 43) by Real Time PCR assay. Dot blot hybridization assay with a sensitivity of 95.5% and specificity of 99.75% could be an ideal choice for routine investigation of a large number of samples in a clinical setting as well as field.